Effects of Functional Groups of Materials on Nonspecific Adhesion

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Effects of Functional Groups of Materials on Nonspecific Adhesion and Chondrogenic Induction of Mesenchymal Stem Cells on Free and Micropatterned Surfaces Bin Cao, Yuanmeng Peng, Xiangnan Liu, and Jiandong Ding ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

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ACS Applied Materials & Interfaces

Effects of Functional Groups of Materials on Nonspecific Adhesion and Chondrogenic Induction of Mesenchymal Stem Cells on Free and Micropatterned Surfaces Bin Cao, Yuanmeng Peng, Xiangnan Liu, Jiandong Ding*

State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200433, China

ACS Paragon Plus Environment


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ABSTRACT: Functional groups of materials are known to affect cell behaviors, yet the corresponding effect on stem cell differentiation are always coupled with that of cell spreading, it is thus unclear whether or not the chemical groups influence cell differentiation directly or via cell spreading indirectly. Herein we used a unique surface patterning technique to decouple the corresponding effects. Mesenchymal stem cells (MSCs) derived from bone marrow were seeded on surfaces coated with alkylthiols with one of 4 functional end groups (-CH3, -OH, -COOH and -NH2) and underwent 9-day chondrogenic induction. The measurements of quartz crystal microbalance with dissipation confirmed less proteins adsorbed from the cell culture media on the neutral -CH3 and -OH surfaces than on the charged -COOH and -NH2 surfaces. The neutral surfaces exhibited less cell spreading and higher extents of chondrogenic differentiation than the charged surfaces, according to the characterizations of immunofluorescence staining and quantitative real-time polymerase chain reaction. We further used a transfer lithography technique to prepare patterned surfaces on nonfouling poly(ethylene glycol) hydrogels to localize single MSCs on microislands with self-assembly monolayers of different alkylthiols, under given microisland areas and thus well-defined spreading areas of cells. While small microislands were always beneficial for chondrogenic induction, we found that the kinds of functional groups had no significant effect on chondrogenic induction under given cell spreading areas, implying that the chemical groups influence cell differentiation only indirectly. Our results hence illustrate that functional groups regulate stem cell differentiation via tuning protein adsorption, then nonspecific cell adhesion and thus cell spreading.

KEYWORDS: Chondrogenic differentiation; Mesenchymal stem cell; Functional group;


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Surface patterning; Self-assembly monolayer; poly(ethylene glycol) (PEG) hydrogel



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1. INTRODUCTION Owing to the complexity of stem cell niche,1-3 one should understand basic principles of cell-material interactions in order to develop biomaterials that can imitate the cell microenvironment. It has been illustrated that various physical and chemical cues of materials influence cell behaviors.4-17 Functional groups, as a particularly important chemical cue, have been found to determine the fate of stem cells, osteoblasts and myoblasts etc.18-28 García et al. 18

reported that –OH and –NH2 terminated surfaces were more beneficial for osteogenesis of

an osteoblast-like cell line MC3T3-E1 than –COOH and –CH3 surfaces. Curran et al.20 carried out a pioneering research to reveal varied in vitro differentiations of mesenchymal stem cells (MSCs) on cell culture glass silanemodified by methyl (–CH3), amino (–NH2), silane (–SH), hydroxyl (–OH) and carboxyl (–COOH). By incorporating functional groups in poly(ethylene glycol) (PEG) hydrogels, Anseth et al.21 found that the phosphate functionalized gel promoted osteogenesis while the t-butyl functionalized gel promoted adipogenesis of MSCs. Tan et al.26 revealed that fibronectin (FN) patterning influenced the integrin orientation demonstrated by super-resolution imaging of human MSCs during the commitment toward the cardiomyogenic lineage. Feng et al.28 modified surfaces by allylamine plasma polymerization, which promoted osteogenic differentiation of human adipose-derived stem cells. Williams et al.23 synthesized polyacrylate with different contents of amine, carboxylic acid and hydroxyl, and discovered that MSCs could undergo chondrogenesis without the addition of transforming growth factor beta (TGF-β) on amine-rich polyacrylate surfaces. In the previous studies, cell differentiation in the material environment of varied functional groups is always accompanied by different cell spreading. It


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remains to be elucidated whether or not the chemical groups influence cell differentiation directly or via cell spreading indirectly. In the present study, we address such a fundamental question, and try to examine stem cell differentiation on surfaces with varied functional groups but with given cell spreading areas. The basic idea is schematically presented in Figure. 1. We employed the combinatory technique of surface patterning and self-assembly monolayer (SAM) to examine such a fundamental question about cell-material interactions. Four kinds of functional groups (-CH3, -OH, -COOH and -NH2) were coated on gold surfaces by a SAM technique. MSCs derived from bone marrow were then seeded on these surfaces and underwent chondrogenic induction in the corresponding medium. We used chondrogenic induction as the differentiation model, not only because it serves as one of standard models,29-30 but also because in vitro chondrogenic differentiation is very important in cartilage tissue engineering.31-33 After 9 days of induction, differentiation extent was obtained by statistics. To further exclude interference factors and explore whether the functional groups affect chondrogenic induction of MSCs directly or not, SAM and surface patterning technique were combined to prepare microislands coated with one kind of 4 groups (-CH3, -OH, -COOH and -NH2), and the fabrication procedures of the micropatterns are schematically illustrated in supplementary Figure S1. Thus, the cell spreading area on different surfaces were fixed, ruling out the interference of other factors such as cell spreading area, cell density, cell-cell contact, and uneven distribution of nutrients.



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Figure 1. Schematic illustration of exploring the effect of functional groups on chondrogenic induction of MSCs.

2. RESULTS 2.1 Self-assembly of functional groups on gold surfaces on matrix of PEG network. In order to keep consistence with the later surface patterning experiments on PEG hydrogels, our SAMs on free surfaces were also generated on the PEG network. So, the gold layer on glass should be transferred to the PEG hydrogel, as schematically presented in Figure 2A.


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Figure 2.

(A) Fabrication of PEG-network based gold surfaces self-assembled with 4

different kinds of functional groups in order to examine the effect of functional groups on MSCs. The terminal functional groups are methyl (-CH3), hydroxyl (-OH), carboxyl (-COOH) or amino (-NH2). (B) Measurement of water contact angles on gold surfaces modified with functional groups with means and standard errors indicated (n = 6). (C) XPS detection of C1s on gold surfaces self-assembled with 4 different functional groups. The binding energies of typical bonds are presented in each graph.

Surfaces with 4 functional groups (-CH3, -OH, -COOH and -NH2) were prepared as described in section 5.1. These surfaces have different surface chemical properties. Wettability of different surfaces was reflected by water contact angel (WCA). The results



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shown in Figure 2B confirmed that the -CH3 surface is hydrophobic while -OH, -COOH and -NH2 surfaces are hydrophilic. Furthermore, X-ray photoelectron spectroscopy (XPS) was applied to detect these 4 surfaces. The binding energies of typical electric orbits of C1s and S2p are indicated in Figure 2C and Figure S2, which demonstrate the successful self-assembly of alkylthiols with 4 different functional groups on gold surfaces. 2.2 Protein adsorption on 4 functional surfaces. By applying quartz crystal microbalance with dissipation (QCM-D), protein adsorption on different functional surfaces can be monitored, with the principle schematically illustrated in Fig. 3A. According to the results presented in Figure 3B, protein adsorption on -CH3, -OH, -COOH and -NH2 surfaces read 614 ± 77, 765 ± 121, 1292 ± 199 and 1383 ± 146 ng/cm2, respectively. So, more proteins were adsorbed on -COOH and -NH2 surfaces than on -CH3 and -OH surfaces.

Figure 3.

(A) Schematic diagram of the principle of QCM-D with definition of the terms

described in section of Materials and Methods. (B) The amount of protein adsorption on surfaces with different functional groups after 9-day chondrogenic induction (n = 3). Significant differences are marked with “**” (p < 0.01) and “***” (p < 0.001). The global test among all of the groups analyzed from One-way ANOVA reads p = 3.08 × 10-4

Effects of Functional Groups of Materials on Nonspecific Adhesion

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